Phi29 Motor Nanopore for Single Molecule Sensing

用于单分子传感的 Phi29 马达纳米孔

• 批准号: 8435052
• 负责人: PEIXUAN GUO
• 金额: $ 8.2万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435052
• 关键词: Phi29; Motor; Nanopore; Single; Molecule

DESCRIPTION (provided by applicant): A wide variety of highly-ordered structures of macromolecules and organized nanomachines exist in biological systems for transporting polynucleic acids and polypeptides. The ingenious design of the bacteriophage phi29 DNA packaging motor is an intriguing element in this category. All linear dsDNA viruses package their genome into a preformed procapsid via an ATP-driving motor involving two nonstructural enzymes or ATPases. The central component of the phi29 motor is the portal connector composed of twelve copies of the protein gp10, which form a dodecamer channel that acts as a pathway for the translocation of double-stranded DNA. With a diameter of 3.6 nm at its narrowest end, it is one of the largest among typical channel proteins that have been incorporated in lipid membranes. Explicit engineering and pore modifications of the phi29 connector are possible due to its available crystal structure, thus inspiring its biomedical applications. The short-term objective of this application is to demonstrate the utility of the lipid-embedded channel of the phi29 DNA packaging motor as a highly sensitive device for characterizing biopolymers at single molecule resolution using electrophysiological assays. The connector will first be reengineered for added functionality and various approaches to modulate the conductance of the connector will be systematically investigated. The dimensions of the pore and charge distributions will then be altered by mutagenesis for specific pore modifications in a controlled fashion to ensure optimal sensing. Finally, the channel will be designed to act as a stochastic biosensor and functionalized with modifying groups to recognize nucleotide bases of double stranded DNA for potential sequencing applications. The long-term objective is to develop the channel into a robust sensing device for detecting pathogens and chemicals at extremely low concentrations for disease diagnosis and environmental monitoring, and to utilize it as a high throughput nanopore-based apparatus for the next generation sequencing of double stranded DNA. The channel can further be used as a tool to assay the structure and function of biological molecules and complexes as well as future avenues for therapeutic DNA loading, packaging, and gene delivery to specific cells for the treatment of cancers, viral infections, and genetics diseases. PUBLIC HEALTH RELEVANCE: The membrane adapted bacteriophage phi29 portal motor nanopore protein will be modified, characterized and developed for highly sensitive detection of pathogens and chemicals at extremely low concentrations as well as for characterizing double-stranded DNA at the single molecule level. The channel can further be developed as a tool for therapeutic DNA packaging and gene delivery to specific cells for the treatment of cancers, viral infections, and genetics diseases.

描述（由申请人提供）： 生物系统中存在多种高度有序的大分子结构和有组织的纳米机器，用于运输多核酸和多肽。噬菌体 phi29 DNA 包装电机的巧妙设计是该类别中一个有趣的元素。所有线性 dsDNA 病毒都通过涉及两种非结构酶或 ATP 酶的 ATP 驱动马达将其基因组包装到预先形成的衣壳中。 phi29 马达的核心部件是由 12 个 gp10 蛋白拷贝组成的门户连接器，形成十二聚体通道，充当双链 DNA 易位的途径。它的最窄端直径为 3.6 nm，是已掺入脂质膜的典型通道蛋白中最大的之一。由于其可用的晶体结构，phi29 连接器的显式工程和孔隙修改是可能的，从而激发了其生物医学应用。 该应用的短期目标是证明 phi29 DNA 包装电机的脂质嵌入通道作为高灵敏度设备的实用性，用于使用电生理测定以单分子分辨率表征生物聚合物。首先将重新设计连接器以增加功能，并将系统地研究调节连接器电导的各种方法。然后，孔的尺寸和电荷分布将通过以受控方式进行特定孔修饰的诱变来改变，以确保最佳传感。最后，该通道将被设计为随机生物传感器，并通过修饰基团进行功能​​化，以识别双链 DNA 的核苷酸碱基，以用于潜在的测序应用。长期目标是将通道开发成强大的传感装置，用于检测极低浓度的病原体和化学物质，以进行疾病诊断和环境监测，并将其用作基于纳米孔的高通量装置，用于下一代双基因测序。搁浅的DNA。该通道还可用作分析生物分子和复合物的结构和功能的工具，以及未来用于治疗性 DNA 加载、包装和基因递送到特定细胞以治疗癌症、病毒感染和遗传疾病的途径。 公共卫生相关性： 膜适应噬菌体 phi29 门运动纳米孔蛋白将被修饰、表征和开发，用于在极低浓度下对病原体和化学物质进行高灵敏度检测，以及在单分子水平上表征双链 DNA。该通道可以进一步开发为治疗性 DNA 包装和基因递送到特定细胞的工具，以治疗癌症、病毒感染和遗传性疾病。